ALK-1 is a type I cell surface receptor for transforming growth factor beta receptor type 1 (TGF-beta-1). Human ALK-1 is a 503 amino acid polypeptide, which includes a signal sequence (amino acids: 1-21), a N-terminal extracellular TGF-beta-1 ligand binding domain or ECD (amino acids: 22-118), a single transmembrane domain (amino acids: 119-141) a regulatory glycine/serine rich (GS) domain (amino acids: 142-202) and a C-terminal a serine-threonine kinase domain (202-492). The amino acid sequence of human ALK-1 disclosed in Attisano et al. Cell, 1993, vol. 75, pp. 671-680 includes Ser at position 172 (Genbank record L17075), while U.S. Pat. No. 6,316,217 claims the amino acid sequence of human ALK-1 with Thr at position 172 (Genbank record NM—000020). ACVRL1 gene encoding a full-length human ALK-1 disclosed in Attisano et al. is commercially available from Invitrogen Inc., Clone ID IOH21048. Although ALK-1 shares 60-80% overall homology with other type I receptors (ALK-2 through ALK-7), ECD of ALK-1 is remarkably divergent from ECDs of other ALK family members. For example, in human, only ECD of ALK-2 is significantly related to ECD of ALK-1 (sharing approximately 25% amino acid identity). U.S. Pat. No. 6,316,217; ten Dijke et al. Oncogene, 1993, vol. 8, pp. 2879-2887; Attisano et al. Cell, 1993, vol. 75, pp. 671-680.
In general, TGF-beta superfamily ligands exert their biological activities via binding to heteromeric receptor complexes of two types (I and II) of serine/threonine kinases. Type II receptors are constitutively active kinases that phosphorylate type I receptor upon ligand binding. In turn, activated type I kinases phosphorylate downstream signaling molecules including the various Smads, which translocate to the nucleus and lead to a transcriptional response. Heldin et al. Nature, 1997, vol. 390, pp. 465-471. In the case of ALK-1, we have shown that Smad1 is specifically phosphorylated and translocates to the nucleus where it directly regulates the expression of the Smad1 responsive genes Id1 and EphB2.
ALK-1 is expressed highly and selectively in endothelial cells and other highly vascularized tissues such as placenta or brain. We have shown by AFFYMETRIX® profiling and real-time RT-PCR that the expression of ALK-1 in endothelial cells highly exceeds the expression of its co-receptors activin type II and endoglin, its ligand TGF-beta-1 or ALK-5. Mutations in ALK-1 are associated with heredity hemorrhagic telangiectasia (HHT), suggesting a critical role for ALK-1 in the control of blood vessel development or repair. Abdalla et al. J. Med. Genet., 2003, vol. 40, pp. 494-502; Sadick et al. Hematologica/The Hematology J., 2005, vol. 90, 818-828. Furthermore, two independent studies of ALK-1 knockout mice provide the key in vivo evidence for ALK-1 function during angiogenesis. Oh et al. Proc Natl Acad Sci USA, 2000, vol. 97, pp. 2626-2631; Urness et al. Nature Genetics, 2000, vol. 26, pp. 328-331.
Angiogenesis is the physiological process involving the formation of new blood vessels from pre-existing vessels and/or circulating endothelial stem cells. This is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state. Hanahan and Folkman, “Patterns and Emerging Mechanisms of the Angiogenic Switch During Tumorigenesis,” Cell, 86(3):353-364, 1996; Carmeliet, “Angiogenesis in Health and Disease,” Nature Medicine, 9(6):653-660, 2003; Bergers and Benjamin, “Tumoreigenesis and the Angiogenic Switch,” Nature Reviews, 3:401-410, 2003. In diseases like cancer, the body loses the ability to maintain balanced angiogenesis. New blood vessels feed diseased tissues, destroy normal tissues, and in the case of some cancers, the new vessels can allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases). Angiogenesis inhibitors, including monoclonal antibodies (mAbs), are a very promising class of drugs targeted against this abnormal process to block or slow tumor growth.
In addition to a role in solid tumor growth and metastasis, other notable conditions with an angiogenic component are, for example, arthritis, psoriasis, neovascular age-related macular degeneration and diabetic retinopathy. Bonnet et al. “Osteoarthritis, Angiogenesis and Inflammation,” Rheumatology, 2005, vol. 44, pp. 7-16; Creamer et al. “Angiogenesis in psoriasis,” Angiogenesis, 2002, vol. 5, pp. 231-236; Clavel et al. “Recent data on the role for angiogenesis in rheumatoid arthritis,” Joint Bone Spine, 2003, vol. 70, pp. 321-326; Anandarajah et al. “Pathogenesis of psoriatic arthritis,” Curr. Opin. Rheumatol., 2004, vol. 16, pp. 338-343; Ng et al. “Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration,” Can. J. Ophthalmol., 2005, vol. 40, pp. 352-368; Witmer et al. “Vascular endothelial growth factors and angiogenesis in eye disease,” Progress in Retinal & Eye Research, 2003, vol. 22, pp. 1-29; Adamis et al. “Angiogenesis and ophthalmic disease,” Angiogenesis, 1999, vol. 3, pp. 9-14.
Anti-angiogenic therapies are expected to be chronic in nature. Accordingly, targets with highly selective endothelial function, such as ALK-1, are preferred to reduce attrition resulting from side effects. Furthermore, given the remarkable divergence of the ALK-1 ECD from ECDs of other ALK family members, mAb raised against the human ALK-1 ECD are expected to selectively target ALK-1. Based on these considerations, a monoclonal antibody against the ALK-1 extracellular domain that may inhibit dimerization with the type II receptor and therefore block Smad1 phosphorylation and the downstream transcriptional response is highly desirable.
R&D Systems, Inc. makes and sells a monoclonal anti-human ALK-1 antibody (Cat. # MAB370) produced from a hybridoma resulting from the fusion of mouse myeloma with B cells obtained from a mouse immunized with purified NS0-derived recombinant human ALK-1 extracellular domain. We have shown that this antibody neither neutralizes the interaction between ALK-1 and TGF-beta-1 nor abrogates Smad1 phosphorylation. Rabbit antisera have been generated against a synthetic peptide corresponding to a part of the intracellular juxtamembrane region of ALK-1 (amino acid residues 145-166), coupled to key-hole limpet haemocyanin (KLH) (U.S. Pat. No. 6,692,925) and against the entire ALK-1 extracellular domain except for the leading sequence (Lux et al., J. Biol. Chem., 1999, vol. 274, pp. 9984-9992). Abdalla et al (Human Mol. Gen., 2000, vol. 9, pp. 1227-1237) report generation of a polyclonal antibody to ALK-1 using a recombinant vaccinia virus construct. R&D Systems, Inc. makes and sells a polyclonal anti-human ALK-1 antibody (Cat. # AF370) produced in goats immunized with purified, NS0-derived, recombinant human ALK-1 extracellular domain.
To date, no fully human monoclonal antibodies to the ECD of ALK-1 have been reported, and no-one has demonstrated the efficacy of any monoclonal antibody to the ECD of ALK-1 in abrogating the ALK-1/TGF-beta-1/Smad1 signaling pathway.